when the cells undergo normal turnover. The inability to detect wild type levels of 
structural proteins in a sample of 1500 infected cells is consistent with either 
mechanism. Because a low level of viral vector was detected in an 
immunocompromised model (xenograft/nude mouse), an issue is whether the vector 
will be recovered in a non-immunocompromised host that is permissive for viral 
replication, e.g., a cotton rat, and primates in the context of the proposed protocol. 
We are addressing this issue in experiments performed as described below. 
IV.B.S.d. Recovery of El deleted adenovirus from cotton rat 
Preliminary experiments in cotton rats indicate that gene transfer can be 
achieved with little inflammation. 300 fi\ of 3 x 10 1 ® pfu/ml Ad5-CB-CFTR and 
Ad5-CMV-lacZ were instilled by midline tracheal incision into two cotton rats (125 
g males). On day 2 the animal was sacrificed and lungs were inflation fixed with 
0.5% glutaraldehyde. Lungs were paraffin embedded and sectioned at 4 pm. X-gal 
staining was predominantly localized in the alveolar regions with some bronchiolar 
staining. No evidence of inflammation was seen. Studies to assess the presence of 
El-deleted adenoviral shedding in cotton rats are underway. 
IV.B.S.e. Absence of recovery of El-deleted adenovirus from baboon 
The aims of this experiment (B210) were to: a) determine if recombinant 
virus is chronically shed; b) assess long and short term toxicity of the Ad5-CB-CFTR 
virus; and c) determine the efficiency and stability of CFTR gene transfer. To 
answer these questions we administered lacZ virus in the left lung and CFTR virus 
in the right lung and subsequently recovered fluid and tissue from each segment via 
a bronchoscope. The data are summarized in Appendix K. 
The animal is a 33 kg male baboon ( Papio cynocephalus) that was 
anesthetized and intubated. Ad5-CMV-lacZ (20 ml, 1 x 10^ pfu/ml) was instilled 
into the posterior segment of the left upper lobe and Ad5-CB-CFTR was instilled 
into the posterior segment of the right upper lobe. After five minutes excess fluid in 
the segments was removed by gentle aspiration. The animal has been evaluated 
three days and two weeks after treatment according to the protocol, which includes: 
physical exam; arterial blood gases; chest X-ray; blood chemistries and 
hematologies; nasal and rectal swabs and blood serum for adenovirus assay; and 
selective bronchoscopy of the two segments with lavage, transbronchi al biopsy, and 
bronchial brushing. 
B210 has tolerated all the procedures well without complications or signs of 
acute toxicity. Lavage from the lacZ segment contained Ad5-CMV-lacZ virus at 
three days; however, this was no longer present at two weeks. Nasal swabs, rectal 
swabs, urine, blood, and lavage from the CFTR segment remained free of 
detectable lacZ virus. The chest X-rays remain normal and arterial blood gases and 
blood chemistries/hematologies remain unchanged (Appendix K). The total 
number of cells recovered in the lavage fluid remained unchanged as did the cell 
differential from the CFTR reconstituted segment; lavage from the lacZ segment 
demonstrated a shift from polys to lymphocytes (Appendix K). 
Cells in the lavage fluid were analyzed for recombinant gene expression 
using X-gal histochemistry for detection of lacZ or immunocytochemistry for 
Recombinant DNA Research, Volume 17 
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